Detachment of Actinobacillus actinomycetemcomitans Biofilm Cells by an Endogenous β-Hexosaminidase Activity

Abstract

When cultured in broth, fresh clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans form tenaciously adherent biofilm colonies on surfaces such as plastic and glass. These biofilm colonies release adherent cells into the medium, and the released cells can attach to the surface of the culture vessel and form new colonies, enabling the biofilm to spread. We mutagenized A. actinomycetemcomitans clinical strain CU1000 with transposon IS903kan and isolated a transposon insertion mutant that formed biofilm colonies which were tightly adherent to surfaces but which lacked the ability to release cells into the medium and disperse. The transposon insertion in the mutant strain mapped to a gene, designated dspB, that was predicted to encode a secreted protein homologous to the catalytic domain of the family 20 glycosyl hydrolases. A plasmid carrying a wild-type dspB gene restored the ability of biofilm colonies of the mutant strain to disperse. We expressed A. actinomycetemcomitans DspB protein engineered to contain a hexahistidine metal-binding site at its C terminus in Escherichia coli and purified the protein by using Ni affinity chromatography. Substrate specificity studies performed with monosaccharides labeled with 4-nitrophenyl groups showed that DspB hydrolyzed the 134 glycosidic bond of ␤-substituted N-acetylglucosamine, which is consistent with the known functions of other family 20 glycosyl hydrolases. When added to culture medium, purified DspB protein, but not heat-inactivated DspB, restored the ability of the mutant strain to release cells and disperse. DspB protein also caused the detachment of cells from preformed biofilm colonies of strain CU1000 grown attached to plastic and the disaggregation of highly autoaggregated clumps of CU1000 cells in solution. We concluded that dspB encodes a soluble ␤-N-acetylglucosaminidase that causes detachment and dispersion of A. actinomycetemcomitans biofilm cells.

Staphylococcal Biofilms

“…In staphylococci, differential expression of the biofilm exopolysacharide PIA might to some degree contribute to biofilm structuring. In contrast, enzymatic degradation of PIA, which appears to occur in other bacteria that express PIA homologs (Kaplan et al 2003), is very likely not present in staphylococci.…”

“…Interestingly, some bacteria produce a PIA-degrading enzyme, which -although not present in staphylococci -can degrade staphylococcal PIA and destroy staphylococcal biofilms (Kaplan et al 2004a). This PIAse, named dispersin B, has first been found in Actinobacillus actinomycetemcomitans, and appears to have potential as an anti-biofilm drug (Kaplan et al 2003). Similarly, although not biofilm-specific, the peptidoglycan-degrading enzyme lysostaphin is being evaluated for therapeutic use against biofilm-forming staphylococci (Wu et al 2003).…”

PslG, a self-produced glycosyl hydrolase, triggers biofilm disassembly by disrupting exopolysaccharide matrix

“…Cellulase has been reported to have some biofilm inhibition activity by partially degrading Psl and Pel in P. aeruginosa [6,22]. A secreted N-acetylglucosaminidase, DspB, has been shown to cause detachment and dispersion of Actinobacillus actinomycetemcomitans biofilm cells [43,44]. However, cellulase and DspB function at millimolar or micromolar concentrations.…”

The Structure- and Metal-dependent Activity of Escherichia coli PgaB Provides Insight into the Partial De-N-acetylation of Poly-β-1,6-N-acetyl-d-glucosamine

“…However, the exact role this domain plays in de-N-acetylation remains unclear. Structurally, the domain is similar to a vast number of glycoside hydrolases including DspB, a well studied glucosaminidase that cleaves PNAG polymers and can disperse biofilms produced by a wide range of bacteria (65)(66)(67). Although glycoside hydrolases typically show activity with artificial substrates such as para-nitrophenyl glycosides (68), no hydrolase activity could be observed for PgaB with para-nitrophenyl GlcNAc or any of the synthetic ␤-1,6-GlcNAc oligomers used in the de-N-acetylation assays (data not shown).…”